Membrane fusion, an essential cellular function, involves poorly understood and very complex membrane interactions. The overall goals of this proposal are to elucidate membrane fusion in a simple lipid system so that this knowledge, along with our new techniques, can be used to help understand biological membrane fusion and to develop liposomes that efficiently deliver therapeutics to cells. While the system has no exact biological counterpart, it allows an unusually detailed examination of bilayer-bilayer merging, a critical stage in biological membrane fusion. The proposal is based on the recent discovery that novel cationic phospholipids form giant vesicles that fuse with giant anionic lipid vesicles. These vesicles can be individually manipulated by voltage gradients under the fluorescence microscope, presenting a unique opportunity to examine bilayer fusion in much greater detail than previously possible. It has now been found that, (a) such bilayer vesicles can fuse without leakage, (b) bilayers can undergo hemifusion (fusion of the two contacting monolayers) in at least three morphologically distinguishable ways, (c) as indicated by fast microspectrofluorometry, full fusion is generally preceded by hemifusion, (d) the vesicle composition (kind of lipid and surface charge density) has a controlling influence on outcomes after contact, i.e., full fusion, one of the hemifusion modes, or simple adhesion and, (e) that the physical properties of cationic phospholipids change in fundamental ways when they are mixed with anionic lipids, in particular, the area occupied by a molecule in a monolayer is reduced and non-lamellar phases (inverted hexagonal or cubic) are generated. It is hypothesized that fusion results from destabilization of the contact region between adherent membranes, either because of charge neutralization and consequent area condensation, or alternatively, because of accumulation of non-lamellar lipids in the contact zone, both of which processes dispose the contacting monolayers to hemifusion and then to full fusion.
The specific aims are: 1. To identify cationic phospholipids that are prone to formation of membrane-destabilizing phases under conditions of vesicle adhesion. 2. To characterize the process of fusion between vesicles of such positive phospholipids with anionic phospholipid vesicles. 3. To apply this information to develop: (a) liposomes that fuse with cells and (b) functional tests of viral membrane components.
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